*Note: The LB top agar/broth and the TM buffer were made based on personal calculations after reviewing Appendex 2: A.2.5 Supplement 40 from Current Protocols in Molecular Biology

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*Note: The LB top agar/broth and the TM buffer were made based on personal calculations after reviewing Appendex 2: A.2.5 Supplement 40 from Current Protocols in Molecular Biology. Amount of LB Top broth was weighed out in grams according to the directions on the bottle. The composition of the top agar was not listed.

1. 0.5L of LB/CaCl2 was made.

1. 0.5L of LB/CaCl2 was made.

Line 139:

Line 145:

A. 1.98g of MgCl2 was added to the 100mL of Tris-Cl that was prepared on 2/13/13

A. 1.98g of MgCl2 was added to the 100mL of Tris-Cl that was prepared on 2/13/13

B. Stock JW-1228 was attempted to be retrieved from -80C freezer, but could not locate.

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C. Instead, JW-1228 + ku (Baij 11/14) from the 4C fridge was used

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D. JW-1228+ku was lightly vortexed because the cells had visibly aggregated to the bottom of the tube.

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E. 1uL of the JW-1228 was put into the 5mL of LB and then put into a 37C incubator overnight.

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2. The TM buffer created on 2/15/2013 was autoclaved

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2/20/2013

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1. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

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A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.

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B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.

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C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.

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D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.

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E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.

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F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

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2. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

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A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.

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B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube and then poured onto a plate. This was done once for each of the two plates.

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C. The plates were then left to harden for about 45 minutes.

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D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates.

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E. Dots 1 and 2 made up the top row, dot 3 was in the middle of the plate, and dots 4 and 5 were located at the bottom of the plate.

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3. The plates were left in the 37C incubator overnight.

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2/21/2013

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1. The plates were retrieved from the incubator at approximately 1150 hours. There were no visible signs of phage plaques. We believe this was due to the fact that the top agar did not solidify as it should have. Solution: Prepare new top agar according to the composition listed in Current Protocols in Molecular Biology, "Top Agar" Unit 1.1

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2. Preparation of the Top Agar

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A. Directions listed for the to make 1 L of Top Agar from Current Protocols in Molecular Biology included:

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10 g tryptone

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5 g Yeast Extract

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5 g NaCl

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7 g Agar

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B. We prepared the Top Agar using the directions on the bottle because its composition was very similar to that listed in the protocol and we felt the difference was negligible.

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3.7 g of Top Agar for 100 ul

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C. The flask used to mix the top agar was labeled "LB Top Agar 12/21/13"

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D. "LB Top Agar 12/21/13 was taken to autoclave

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Autoclave set to program 9 for liquid autoclaving of 15 minutes.

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3. Re-preparation of Top Agar

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A. After further examination of the Top agar a more suitable batch was made, that more closely matched the directions listed in part A of step 2 of 2/21/2013's intended ingredients.

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B. 2g of a mixture that had similar concentrations of the ingredients tryptone(peptone instead), yeast extract, and NaCl. The concentrations of the NaCl was twice as what it normally is intended to be, but it was thought that this would not affect results.

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C. 0.7g of agar was added to the mixture in part B and then the entire mixture was added to 100mL of water and stirred until it was relatively clear.

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2/22/2013

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1. The re-prepared Top Agar was autoclaved on setting 9 and left to cool.

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2. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

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A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.

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B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.

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C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.

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D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.

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E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.

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F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

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3. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

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A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.

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B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube and then poured onto a plate. This was done once for each of the two plates.

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C. The plates solidified relatively quickly, about less than a minute.

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D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates.

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E. Dots 1 and 2 made up the top row, dot 3 was in the middle of the plate, and dots 4 and 5 were located at the bottom of the plate.

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4. The plates were left in the 37C incubator overnight.

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2/23/2013

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1. Retrieved plates from incubator left over night. Looks like there is some clearing around number 3 on one plate and on the other plate the gel is damaged, but there appears to be some clearing around some of the damage and dot number 4.

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2/24/2013

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1. Obtained previously made top agar 2/21/2013 and put it in the microwave for about 35 seconds, in 5 and 10 second intervals until liquified.

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2. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

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A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.

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B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.

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C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.

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D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.

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E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.

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F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

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3. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

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A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.

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B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube and then poured onto a plate. This was done once for each of the two plates.

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C. The plates solidified relatively quickly, about less than a minute.

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D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates.

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E. Dots 1 and 2 made up the top row, dot 3 was in the middle of the plate, and dots 4 and 5 were located at the bottom of the plate.

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4. The plates were left in the 37C incubator overnight.

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2/25/2013

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1. Obtained the plates made on 2/24/2013

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A. Observed similar clearing to the last set of plates where it looks like there is some discoloration in the agar defined by everything slightly above dot 3 being less opaque than everything below it.

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B. Moreover, there were a few well defined plaques that existed between dots 1 and 3, with a few other plaques scattered around other dots.

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C. Calculations will not be made until it can be confirmed that the clearing is/is not due to the presence of phage in that particular area.

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2. Obtained previously made top agar 2/21/2013 and put it in the microwave for about 45 seconds, in 5 and 7 second intervals until liquified.

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3. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

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A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.

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B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.

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C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.

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D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.

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E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.

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F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

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4. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

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A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.

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B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube, vortexted, and then poured onto a plate. This was done once for each of the two plates.

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C. The plates solidified relatively quickly, about less than a minute.

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D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates.

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E. Dots 1 and 2 made up one plate divided down the middle and dots 3, 4, and 5 were located on another plate in a peace sign like configuration.

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5. The plates were left in the 37C incubator overnight.

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2/26/2013

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1. Plates were obtained from the incubator.

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A. Plates with the dilutions of 10^-1 and 10^-3 exhibited a lot of plaques. In the case of the half of the plate with the 10^-1 dilution there were large regions of the top agar missing possibly suggesting that there was a lot of phage activity to the point that it was large combined plaques.

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B. In the case of the plates with the dilutions of 10^-5, 10^-7, 10^-8, there was a relevantly large population of phage around the 10^-5 dilution.

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2. 1uL of the JW-1228 was put into the 5mL of LB and then put into a 37C incubator overnight.

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2/27/2013

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1. Obtained previously made top agar 2/21/2013 and put it in the microwave for about 45 seconds, in 5 and 7 second intervals until liquified.

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2. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

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A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.

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B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.

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C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.

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D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.

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E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.

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F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

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3. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

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A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.

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B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube, vortexted, and then poured onto a plate. This was done once for each of the two plates.

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C. The plates solidified relatively quickly, about less than a minute.

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D. After the plates were somewhat solidified plates, 100uL from each of the 5 tubes were dotted and then spread onto the plates. The plates were numbered in accordance to the dilution of phage present on them.

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4. The plates were left in the 37C incubator overnight.

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2/28/2013

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1. Plates were retrieved from the incubator and observations were made.

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A. All 5 plates were taken out of the incubator at 11:50am.

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B. It appeared that there was top agar damage on each plate that may or may not have been due to the phage.

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C. While there were a lot of plaques as expected of the addition of 100uL of phage dilution to each plate, an accurate number might not have been determinable because of the excessive damage to the top agar.

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D. Plates were put in the 4C fridge over night for future examinations.

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3/01/2013

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1. Observations of the plates made on 2/26/2013 were reassessed and then calculations were made using the formula: (Number of Plaques)/(Dilution Factor * Volume (in mL) = PFU/mL.

E. Plaques observed for dot 1 were not distinguishable due to the large amounts of clearing observed, so calculations could not be accurately made.

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3/11/13

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P1 Transduction

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1. Add 5 ml of LB broth to 14 ml culture tube at 3:25 pm

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2. Added FMJ39 Competent E.coli to the culture tube

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a. The top of the microcentrifuge tube was scraped with a pipet and the pipet as long as the cell contents was released into the culture tube

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3. The culture tube named "FMJ 39 Baij" was set into the incubator to incubate till 12 noon on 03/12/13

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4. The tubes were accidently removed at 6:30pm 03/11/13 and put into the fridge. On 03/12/13 these tubes were put back in the incubator at 12:15pm and will be retrieved at 8:15am 03/13/13.

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a. Possible contamination when removed early due to taking the cultures out and attempting to count them with a hemocytometer.

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3/12/13

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1. Agar plates were made.

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A. 500mL of agar was made. 3.5g of agar and 10g of a mixture containing tryptone(peptone instead), yeast extract, and NaCl was added to 500mL of water.

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B. The solution was stirred and then autoclaved at 12:39pm and retrieved at about 1:45pm and let cool.

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C. Once cool the agar and plates were brought to the fume hood that was sprayed down with 70% alcohol solution.

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D. The plates were filled about halfway with the agar solution, swirled for evenness, and then let to solidify.

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E. Once all the plates were poured they were put back in their bag and labeled "CH agar 03/13/2013" and put in the walk in freezer.

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3/13/13

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1. "FMJ 39 Baij" was removed from the incubator and placed in fridge at 8:09 am. There were visible signs of a large amount of microbial growth including the broth looking very turbid.

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3/14/2013

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1. P1 salts were made.

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A. 0.147g of CaCl2 (10mM) and 0.124g of MgSO4 (5mM) were added to 100mL of water and then autoclaved on setting 9.

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2. Used flasks and bottles were autoclaved.

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3/15/13

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1. Transformed FMJ Culture was plated at concentration of 10 ml and 100 ml of kanamycin (two plates) along with a control plate that only contained cells and not any of the antibiotic.

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2. The transformed culture plates are to be scored on 3/16/13.

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3/16/13

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1. The plates were scored.

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2. The control plate at bacterial growth, which was expected. The control contained pure FMJ strain with no Kanamycin.

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3. All other plates (1 10 ml, and 2 100 ml) showed bacterial growth.

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4. The cultures were replated with 20 microliters of Kanamycin to be scored on 3/18

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A. Kanamycin was added to ensure that the cultures retained antibiotic resistance.

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3/18/13

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1. The plates labeled Transduced FMJ + KM were removed from the incubator at 3:30 pm and scored.

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2. There a appears to be a large amount of microbial growth toward the edges of the plate where there was a smaller concentration of KM. There was no sign of microbial growth in the middle of the plate where the largest amount of KM would be present. There may be a false positive present since the Transduced FMJ was mixed with KM and then plated on LB plates instead of selective plates.

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3. The experiment should be redone using KM selected plates to fix the above possible error.

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3/21/13

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1. A colony of cells were chosen on the 100ml of kanamycin plate. Two designated areas containing a high number of colonies was marked and then 5uL of kanamycin was pipetted onto that area in hopes that the presence of these cells did not represent a false positive.

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3/22/13

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1. Plates were observed and there was not any change. This is not indicative of anything other than the colonies present are resistant to kanamycin

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05/07/2013

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1. Added 50uL of JW 1228-1 from stock "Baij 11/4" from the 4 C fridge to 5mL of LB "8/27" and put in the incubator over night. Put in at 12:05pm

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05/08/2013

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1. Removed JW 1228 bacteria from incubator at 12:25pm and put into the 4C fridge

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05/15/2013

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1. Cells were removed from the 4C fridge.

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2. Steps were followed from the Promega MagaZorb kit up, with the only deviation being that 1xPBS buffer was used instead of 10xPBS buffer.

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3. Due to time constraints the last step that was performed was putting in 500mL of binding buffer.

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4. If the products from these steps are salvagable, they are in a tube labelled "Xtract CH" in the 4C fridge.

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05/19/2013

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Submission Team's Progress - Norman Luong

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Our objective is to insert isolated coding sequences for AdhE (AKA MphF) and FO into the plasmid backbone pSB1C3. Through Fall 2012 Team's efforts, AdhE and FO have already been amplified via PCR and gel purification. The problem that they faced and we attempted to solve during Spring 2013 was to amplify the backbone via bacterial transformation followed by DNA extraction. We were successful in extracting enough DNA to see a set of three bands, which is characteristic of the different conformations that circular DNA can take. However, after gel extraction and restriction digestion of the band that we believed to represent to the supercoiled conformation, we did not see any DNA in the following gel. Thus, we turned to PCR to amplify the backbone, which conveniently also gives us a linear product as long as the extension time is set properly. We were able to run a PCR using pSB1C3 extracted from transformed bacteria before the semester ended. The next step is to analyze and purify the product by gel electrophoresis, then prepare the insert and backbone for ligation by doing restriction digest (RD). Despite the products being linear, RD is necessary in order to create sticky ends for ligation. The insert must be RD'd by EcoRI and SpeI, and the backbone must be RD'd by EcoRI and XbaI (see http://partsregistry.org/Assembly:Standard_assembly). No measureable difference in gel mobility is expected between the RD and pre-RD samples. This process must be done separately for both MphF and FO. Once the insert and backbone are ligated, the product must be analyzed by UV/Vis spec (Nanodrop) to ensure that the product meets the minimum concentration of 25 ng/uL.

Current revision

Description/Abstract

Energy production in modern society relies primarily on fossil fuels. However, fossil
fuels are a limited resource and their use generates pollution. Our goal is to create
a sustainable and clean energy source to fossil fuel. Hydrogen gas is considered a
clean alternative energy source. To achieve this goal, we engineered the
fermentation pathway in E. coli FMJ39 for H2 gas production. This strain is mutated
for the ldhA and pflB genes, involved in mixed acid fermentation. Also, we will
delete gene adhE by P1 phage generalized transduction. Additionally, we cloned
the genes of dark fermentation Acetaldehyde dehydrogenase (MphF) from
Methylophilales bacterium, and Ferredoxin oxidoreductase (FO) from E. coli W and
transformed E. coli FMJ39, creating a new pathway for optimal production of
hydrogen gas. Producing such an alternative fuel will address the rising issues of a
dwindling fossil fuel supply, and mitigate its demand as a future primary fuel source.

1. Overnight culture did not grow. Possible reason is: The JW1228-1 culture taken from the 4°C refrigerator was old and was probably no longer living. To address this possibility we will redo the procedure using frozen JW1228-1 stock.
2. Prepared new overnight culture with frozen JW1228-1 glycerol stock (following the procedure carried out on 10/13/12)

5. JW1228-1 overnight culture prepared for P1 phage solution to be incubated until 11:55 on 11/15/12

6. 20ul of P1 phage was added to JW1228-1/P1 solution after incubation. It will be incubated until 11/15/12 at 3 pm

7. JW1228-1/P1 culture was checked at 3 pm and there was a large amount of turbidity, which is opposite to what we should have seen. This means that potentially the P1 phage did not infect the JW1228-1 cells and instead, the JW1228-1 cells continued to grow through incubation. Due to this observation the JW1228-1/P1 culture was left to incubate until 4:30 pm on 11/15/12.

8. JW1228-1/P1 culture was removed from the incubator at 4:36 pm on 11/15/12. The JW1228-1/P1 culture appeared less turbid then when checked at 3 pm however, still more turbid then expected due to P1 phage activity.

9. Drops of chloroform were added to the JW1228-1/P1 culture and 3 ml of solution was centrifuged in each culture tube. Only 2 ml of supernatant was available from each 35 ml centrifuge tube. The supernatant was kept in 1 ml tubes and four tubes were collected.

10. The 1 ml tubes are labeled as CH AB 11/15 and kept in the 4°C refrigerator.

Note: The LB top agar/broth and the TM buffer were made based on personal calculations after reviewing Appendex 2: A.2.5 Supplement 40 from Current Protocols in Molecular Biology. Amount of LB Top broth was weighed out in grams according to the directions on the bottle. The composition of the top agar was not listed.

1. 0.5L of LB/CaCl2 was made.

A. 19.99g of LB agar was added to 500mL of water, to bring the total content of the Erlenmeyer flask up to approximately 500mL.
B. 147g/mole CaCl2 * (0.0025 mole/ L) * 0.5L = 0.18375g CaCl2
C. 0.19g of CaCl2 was added to the 0.5 solution
D. The 0.5L of LB/CaCl2 was separated into two Erlenmeyer flasks, each with about 0.25L in each.

2. 0.1L of Top agar LB/CaCl2 was made.

A. 2g of LB broth was added to 100mL of water, to bring the content of the Erlenmeyer flask up to approximately 100mL
B.147g/mole CaCl2 * (0.0025 mole/ L) * 0.1L = 0.3675g CaCl2

3. Both were then autoclaved on the 15 minute cycle (setting 9).

A. Once out, the two 250mL flasks containing the LB/CaCl2 were put onto ice to cool.
B. The top agar solution, once cooled, was labeled and left on the bench for future use.
C. The solution is labeled as "CaCl2 Top agar 2/13/12"

4. The two 250mL flasks containing LB/Cacl2 were taken to a fume hood that was previously sterilized with 37% alcohol solution, for plates to be made.

A. The LB/CaCl2 solution was poured onto each plate and then the plates were swirled to allow for the solution to be evenly distributed over each plate.
B. In total about 30 of these plates were left to sit with their tops off in the hood for 15-25 minutes to harden.
C. Once the plates had hardened they were put back into the bag in which they came, labeled with a black and blue line, and then put into the walk in refrigerator.
D. After about an hour of being left to harden, approximately 16 plates did not solidify and were then disposed of. This could be due to inadequate mixing of the original agar solution.

5. 100 mM Tris-Cl was prepared for a TM buffer.

A. 12.1g of Tris base was added to 100mL of water and stirred until the solution became clear.
B. 5.3mL of HCl was added to the solution to bring the solution to a final pH of 7.38
C. The solution was labeled and left on the bench for future use.

2/15/2013

1. Completion of 100mL of TM buffer.

A. 1.98g of MgCl2 was added to the 100mL of Tris-Cl that was prepared on 2/13/13
B. The concentrations of the Tris-Cl and the MgCl2 is 100mM each.

2/19/2013 (12:05 pm)

1. Bacteria for future use were cultured

A. 5mL of LB (prepared on 8/27 from lab bench) was placed into 14 ml round bottom tube.
B. Stock JW-1228 was attempted to be retrieved from -80C freezer, but could not locate.
C. Instead, JW-1228 + ku (Baij 11/14) from the 4C fridge was used
D. JW-1228+ku was lightly vortexed because the cells had visibly aggregated to the bottom of the tube.
E. 1uL of the JW-1228 was put into the 5mL of LB and then put into a 37C incubator overnight.

2. The TM buffer created on 2/15/2013 was autoclaved

2/20/2013

1. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.
B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.
C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.
D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.
E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.
F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

2. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.
B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube and then poured onto a plate. This was done once for each of the two plates.
C. The plates were then left to harden for about 45 minutes.
D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates.
E. Dots 1 and 2 made up the top row, dot 3 was in the middle of the plate, and dots 4 and 5 were located at the bottom of the plate.

3. The plates were left in the 37C incubator overnight.

2/21/2013

1. The plates were retrieved from the incubator at approximately 1150 hours. There were no visible signs of phage plaques. We believe this was due to the fact that the top agar did not solidify as it should have. Solution: Prepare new top agar according to the composition listed in Current Protocols in Molecular Biology, "Top Agar" Unit 1.1

2. Preparation of the Top Agar

A. Directions listed for the to make 1 L of Top Agar from Current Protocols in Molecular Biology included:
10 g tryptone
5 g Yeast Extract
5 g NaCl
7 g Agar
B. We prepared the Top Agar using the directions on the bottle because its composition was very similar to that listed in the protocol and we felt the difference was negligible.
3.7 g of Top Agar for 100 ul
C. The flask used to mix the top agar was labeled "LB Top Agar 12/21/13"
D. "LB Top Agar 12/21/13 was taken to autoclave
Autoclave set to program 9 for liquid autoclaving of 15 minutes.

3. Re-preparation of Top Agar

A. After further examination of the Top agar a more suitable batch was made, that more closely matched the directions listed in part A of step 2 of 2/21/2013's intended ingredients.
B. 2g of a mixture that had similar concentrations of the ingredients tryptone(peptone instead), yeast extract, and NaCl. The concentrations of the NaCl was twice as what it normally is intended to be, but it was thought that this would not affect results.
C. 0.7g of agar was added to the mixture in part B and then the entire mixture was added to 100mL of water and stirred until it was relatively clear.

2/22/2013

1. The re-prepared Top Agar was autoclaved on setting 9 and left to cool.

2. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.
B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.
C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.
D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.
E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.
F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

3. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.
B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube and then poured onto a plate. This was done once for each of the two plates.
C. The plates solidified relatively quickly, about less than a minute.
D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates.
E. Dots 1 and 2 made up the top row, dot 3 was in the middle of the plate, and dots 4 and 5 were located at the bottom of the plate.

4. The plates were left in the 37C incubator overnight.

2/23/2013

1. Retrieved plates from incubator left over night. Looks like there is some clearing around number 3 on one plate and on the other plate the gel is damaged, but there appears to be some clearing around some of the damage and dot number 4.

2/24/2013

1. Obtained previously made top agar 2/21/2013 and put it in the microwave for about 35 seconds, in 5 and 10 second intervals until liquified.
2. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.
B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.
C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.
D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.
E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.
F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

3. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.
B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube and then poured onto a plate. This was done once for each of the two plates.
C. The plates solidified relatively quickly, about less than a minute.
D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates.
E. Dots 1 and 2 made up the top row, dot 3 was in the middle of the plate, and dots 4 and 5 were located at the bottom of the plate.

4. The plates were left in the 37C incubator overnight.

2/25/2013

1. Obtained the plates made on 2/24/2013

A. Observed similar clearing to the last set of plates where it looks like there is some discoloration in the agar defined by everything slightly above dot 3 being less opaque than everything below it.
B. Moreover, there were a few well defined plaques that existed between dots 1 and 3, with a few other plaques scattered around other dots.
C. Calculations will not be made until it can be confirmed that the clearing is/is not due to the presence of phage in that particular area.

2. Obtained previously made top agar 2/21/2013 and put it in the microwave for about 45 seconds, in 5 and 7 second intervals until liquified.
3. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.
B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.
C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.
D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.
E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.
F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

4. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.
B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube, vortexted, and then poured onto a plate. This was done once for each of the two plates.
C. The plates solidified relatively quickly, about less than a minute.
D. After the plates were somewhat solidified 5 dots, 10uL of each of the 5 tubes were dotted onto the plates. The dots were numbered in accordance to the number of test tube and their location was denoted by the location of the label written on the plates.
E. Dots 1 and 2 made up one plate divided down the middle and dots 3, 4, and 5 were located on another plate in a peace sign like configuration.

5. The plates were left in the 37C incubator overnight.

2/26/2013

1. Plates were obtained from the incubator.

A. Plates with the dilutions of 10^-1 and 10^-3 exhibited a lot of plaques. In the case of the half of the plate with the 10^-1 dilution there were large regions of the top agar missing possibly suggesting that there was a lot of phage activity to the point that it was large combined plaques.
B. In the case of the plates with the dilutions of 10^-5, 10^-7, 10^-8, there was a relevantly large population of phage around the 10^-5 dilution.

2. 1uL of the JW-1228 was put into the 5mL of LB and then put into a 37C incubator overnight.

2/27/2013

1. Obtained previously made top agar 2/21/2013 and put it in the microwave for about 45 seconds, in 5 and 7 second intervals until liquified.
2. Serial ten-fold dilutions were made with the phage stock and the TM buffer.

A. Five 1mL tubes were labeled 1 to 5 to represent different concentrations of phage in TM buffer.
B. Tube 1 contained 900uL of TM buffer and 100uL of phage stock for a dilution of 10^-1, after the addition of the phage the tube was vortexed.
C. Tube 2 contained 990uL of TM buffer and 10uL of Tube 1 was added to the tube for a dilution of 10^-3, after the addition of Tube 1, Tube 2 was vortexed.
D. Tube 3 contained 990uL of TM buffer and 10uL of Tube 2 was added to the tube for a dilution of 10^-5, after the addition of Tube 2, Tube 3 was vortexed.
E. Tube 4 contained 990uL of TM buffer and 10uL of Tube 3 was added to the tube for a dilution of 10^-7, after the addition of Tube 3, Tube 4 was vortexed.
F. Tube 5 contained 900uL of TM buffer and 100uL of Tube 4 was added to the tube for a dilution of 10^-8, after the addition of Tube 4, Tube 5 was vortexed.

3. A lawn of JW-1228 cells cultured overnight on 2/19/2013 were plated and dots of phage at different dilutions were put on top of this plate.

A. Two LB/CaCl2 plates from the plates made on 2/13/2013 were taken out of the walk-in freezer.
B. 250uL of the JW-1228 cells cultured the night before along with 2.5mL of the LB top agar made on 2/13/2013 was mixed together in a test tube, vortexted, and then poured onto a plate. This was done once for each of the two plates.
C. The plates solidified relatively quickly, about less than a minute.
D. After the plates were somewhat solidified plates, 100uL from each of the 5 tubes were dotted and then spread onto the plates. The plates were numbered in accordance to the dilution of phage present on them.

4. The plates were left in the 37C incubator overnight.

2/28/2013

1. Plates were retrieved from the incubator and observations were made.

A. All 5 plates were taken out of the incubator at 11:50am.
B. It appeared that there was top agar damage on each plate that may or may not have been due to the phage.
C. While there were a lot of plaques as expected of the addition of 100uL of phage dilution to each plate, an accurate number might not have been determinable because of the excessive damage to the top agar.
D. Plates were put in the 4C fridge over night for future examinations.

3/01/2013

1. Observations of the plates made on 2/26/2013 were reassessed and then calculations were made using the formula: (Number of Plaques)/(Dilution Factor * Volume (in mL) = PFU/mL.

1. Add 5 ml of LB broth to 14 ml culture tube at 3:25 pm
2. Added FMJ39 Competent E.coli to the culture tube
a. The top of the microcentrifuge tube was scraped with a pipet and the pipet as long as the cell contents was released into the culture tube
3. The culture tube named "FMJ 39 Baij" was set into the incubator to incubate till 12 noon on 03/12/13
4. The tubes were accidently removed at 6:30pm 03/11/13 and put into the fridge. On 03/12/13 these tubes were put back in the incubator at 12:15pm and will be retrieved at 8:15am 03/13/13.
a. Possible contamination when removed early due to taking the cultures out and attempting to count them with a hemocytometer.

3/12/13

1. Agar plates were made.

A. 500mL of agar was made. 3.5g of agar and 10g of a mixture containing tryptone(peptone instead), yeast extract, and NaCl was added to 500mL of water.
B. The solution was stirred and then autoclaved at 12:39pm and retrieved at about 1:45pm and let cool.
C. Once cool the agar and plates were brought to the fume hood that was sprayed down with 70% alcohol solution.
D. The plates were filled about halfway with the agar solution, swirled for evenness, and then let to solidify.
E. Once all the plates were poured they were put back in their bag and labeled "CH agar 03/13/2013" and put in the walk in freezer.

3/13/13

1. "FMJ 39 Baij" was removed from the incubator and placed in fridge at 8:09 am. There were visible signs of a large amount of microbial growth including the broth looking very turbid.

3/14/2013

1. P1 salts were made.

A. 0.147g of CaCl2 (10mM) and 0.124g of MgSO4 (5mM) were added to 100mL of water and then autoclaved on setting 9.

2. Used flasks and bottles were autoclaved.

3/15/13

1. Transformed FMJ Culture was plated at concentration of 10 ml and 100 ml of kanamycin (two plates) along with a control plate that only contained cells and not any of the antibiotic.
2. The transformed culture plates are to be scored on 3/16/13.

3/16/13

1. The plates were scored.
2. The control plate at bacterial growth, which was expected. The control contained pure FMJ strain with no Kanamycin.
3. All other plates (1 10 ml, and 2 100 ml) showed bacterial growth.
4. The cultures were replated with 20 microliters of Kanamycin to be scored on 3/18

A. Kanamycin was added to ensure that the cultures retained antibiotic resistance.

3/18/13

1. The plates labeled Transduced FMJ + KM were removed from the incubator at 3:30 pm and scored.
2. There a appears to be a large amount of microbial growth toward the edges of the plate where there was a smaller concentration of KM. There was no sign of microbial growth in the middle of the plate where the largest amount of KM would be present. There may be a false positive present since the Transduced FMJ was mixed with KM and then plated on LB plates instead of selective plates.
3. The experiment should be redone using KM selected plates to fix the above possible error.

3/21/13

1. A colony of cells were chosen on the 100ml of kanamycin plate. Two designated areas containing a high number of colonies was marked and then 5uL of kanamycin was pipetted onto that area in hopes that the presence of these cells did not represent a false positive.

3/22/13

1. Plates were observed and there was not any change. This is not indicative of anything other than the colonies present are resistant to kanamycin

05/07/2013

1. Added 50uL of JW 1228-1 from stock "Baij 11/4" from the 4 C fridge to 5mL of LB "8/27" and put in the incubator over night. Put in at 12:05pm

05/08/2013

1. Removed JW 1228 bacteria from incubator at 12:25pm and put into the 4C fridge

05/15/2013

1. Cells were removed from the 4C fridge.
2. Steps were followed from the Promega MagaZorb kit up, with the only deviation being that 1xPBS buffer was used instead of 10xPBS buffer.
3. Due to time constraints the last step that was performed was putting in 500mL of binding buffer.
4. If the products from these steps are salvagable, they are in a tube labelled "Xtract CH" in the 4C fridge.

05/19/2013
Submission Team's Progress - Norman Luong
Our objective is to insert isolated coding sequences for AdhE (AKA MphF) and FO into the plasmid backbone pSB1C3. Through Fall 2012 Team's efforts, AdhE and FO have already been amplified via PCR and gel purification. The problem that they faced and we attempted to solve during Spring 2013 was to amplify the backbone via bacterial transformation followed by DNA extraction. We were successful in extracting enough DNA to see a set of three bands, which is characteristic of the different conformations that circular DNA can take. However, after gel extraction and restriction digestion of the band that we believed to represent to the supercoiled conformation, we did not see any DNA in the following gel. Thus, we turned to PCR to amplify the backbone, which conveniently also gives us a linear product as long as the extension time is set properly. We were able to run a PCR using pSB1C3 extracted from transformed bacteria before the semester ended. The next step is to analyze and purify the product by gel electrophoresis, then prepare the insert and backbone for ligation by doing restriction digest (RD). Despite the products being linear, RD is necessary in order to create sticky ends for ligation. The insert must be RD'd by EcoRI and SpeI, and the backbone must be RD'd by EcoRI and XbaI (see http://partsregistry.org/Assembly:Standard_assembly). No measureable difference in gel mobility is expected between the RD and pre-RD samples. This process must be done separately for both MphF and FO. Once the insert and backbone are ligated, the product must be analyzed by UV/Vis spec (Nanodrop) to ensure that the product meets the minimum concentration of 25 ng/uL.